Two-photon microscopy (TPM) can provide high resolution and high contrast, even when imaging in scattering tissue. TPM can be used to resolve single cells, such as neurons and subneuronal structures, up to one millimeter deep in tissue. TPM has been used extensively to measure dynamic processes, such as calcium dynamics, in populations of neurons in the intact brain, even during animal behavior. Up to several hundred neurons have been imaged simultaneously, but largely limited to fields of view on the order of half a millimeter within single brain areas.
In point-scanning laser scanning microscopy (LSM), a focused laser is moved over a sample in three dimensions. Laser scanning can be achieved relatively easily in the plane (x-y plane) perpendicular to the optical axis of the scanned beam, but is much more challenging along the optical axis (z), parallel to the direction of the beam is much more challenging. In many microscopes to scan the beam along the optical axis, the objective is physically moved to change the z-location of the focus, but this involves moving a large mass and therefore is relatively slow, limiting the types of images that can be acquired and the types of microscopes that can be practically designed. Alternatively, a stage that supports the sample can be moved along the optical axis while the location of objective remains fixed, but this also involves moving a large mass.
Several applications would benefit from microscopes with much larger fields of view while retaining cellular resolution. Even relatively simple animal behavior involves multiple brain regions, which are often non-contiguous. Probing the interaction of these spatially separated requires imaging both brain areas nearly simultaneously. In primates, cortical areas of interest are often separated by several millimeters across a gyms. Other applications include tracking cellular structures in developing embryos. Imaging multiple brain areas simultaneously is not possible using standard microscopes. High-resolution microscopes have small fields of view, whereas large field-of-view microscopes do not have cellular resolution. The advent of sensitive protein indicators for neural activity (e.g. calcium indicators) and transgenic animals expressing these indicators opens up the possibility of mesoscale imaging. Note that mesoscale microscopy is defined as imaging with cellular resolution and fields of view spanning multiple brain areas or entire organisms (several millimeters).